Analysis of the S-2 subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids

We have explored the specificity of the S-2 subsite of recombinant cysteine proteinases from Leishmania mexicana (CPB2.8 Delta CTE) and from Trypanosoma cruzi (cruzain) employing a series of fluorogenic substrates based on the peptide Bz-F-R-MCA, in which Bz is the benzoyl group and the Phe residue has been substituted for by Arg, His and non-natural basic amino acids that combine a basic group with an aromatic or hydrophobic group at the side chain: 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinylalanine (Ppa). 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). Bz-F-R-MCA was hydrolyzed well by CPB2.8 Delta CTE and cruzain, but all the substitutions of Phe resulted in less susceptible substrates for the two enzymes. CPB2.8 Delta CTE has a restricted specificity to hydrophobic side chains as with cathepsin L. However, the peptides with the residues Amf and Ama presented higher affinity to CPB2.8ACTE, and the latter was an inhibitor of the enzyme. Although, cruzain accepts basic as well as hydrophobic residues at the S2 Subsite, it is more restrictive than cathepsin B and no inhibitor was found amongst the examined peptides. (C) 2001 Elsevier Science B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilUniv Glasgow, Inst Biomed & Life Sci, Div Infect & Immun, Glasgow G12 8QQ, Lanark, ScotlandUniv Glasgow, Anderson Coll, Wellcome Ctr Mol Parasitol, Glasgow G11 6NU, Lanark, ScotlandUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilWeb of Scienc